Chloral-free dca in oligonucleotide synthesis

ABSTRACT

A process of manufacturing oligonucleotides includes a 5′-deblocking step in which the 5-blocking group is removed with dichloroacetic acid that is essentially free of chloral. The process is useful for making oligonucleotides that are substantially free of chloral adducts.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. provisional application60/369,295, filed on Apr. 1, 2002, which is explicitly incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention is directed to the field of oligonucleotidesynthesis. In particular, the present invention is directed to improvedoligonucleotide synthetic methods, whereby improved oligonucleotidecharacteristics are obtained.

BACKGROUND OF THE INVENTION

Oligomeric compounds having the ability to specifically bind natural andsynthetic polynucleotides have numerous uses in analytical methods fordetection, identification, and quantification of polynucleotides, asprimers and probes for amplifying genes and gene products (e.g. thepolymerase chain reaction, PCR), in target validation studies and astherapeutics. Oligomeric compounds such as oligonucleotide DNA and RNAhave been used successfully to detect natural polynucleotides andpolynucleotide products on so-called biochips. Oligomeric compounds canalso be used as primers and probes for taq-polymerase in PCR. Variousoligonucleotide compounds and derivatives thereof have been successfullyemployed in gene-silencing, both in vitro and in vivo. Sucholigonucleotide compounds and their derivatives include so-calledantisense compounds—oligomers capable of specifically binding a gene orgene product, and either directly or indirectly effecting silencing ofthe gene.

Antisense therapeutics have shown great promise. Antisense therapeuticsmodulate protein activities by attenuating the concentration ofoligonucleotides, especially RNA, involved in protein synthesis. This isin contrast to conventional therapeutic methods, which seek to modulateprotein activities by direct interaction between putative drugs andproteins.

In general, antisense methods involve determining the sequence of acoding oligonucleotide (e.g. mRNA) that encodes for a certain protein(sense strand), developing a relatively short oligomer that selectivelybinds to the sense strand, and introducing the oligomer into theintracellular environment. Antisense methods can predictably silencegene expression through a variety of mechanisms. In one such mechanism,Translation Arrest, the antisense strand blocks translation bycompetitively binding to the sense strand of mRNA. In another mechanism,an antisense strand containing a stretch of DNA (e.g. phosphorothioateDNA) binds to the sense strand, whereby the DNA-RNA hybrid is recognizedby RNAse H, an endonuclease that selectively cleaves the DNA-RNA hybrid,thereby reducing intracellular RNA levels. Another methodology involvesthe interaction between small double stranded RNA oligomers and mRNA. Insuch mechanisms, interaction between the RISC complex, the antisensestrand of the small double stranded RNA and intracellular mRNA resultsin cleavage and degradation of the mRNA.

As antisense molecules have become accepted as therapeutic anddiagnostic agents, the need to produce oligonucleotides in largequantities, at higher purity, and at decreased per unit cost hasincreased as well. The most commonly used antisense compounds to datehave been phosphodiester oligonucleotides, phosphorothioateoligonucleotides and second generation oligonucleotides having one ormore modified ribosyl sugar units, and more recently, ribosyl sugarunits. The methods for making these three types of antisense oligomersare roughly similar, and include the phosphotriester method, asdescribed by Reese, Tetrahedron 1978, 34, 3143; the phosphoramiditemethod, as described by Beaucage, in Methods in Molecular Biology:Protocols for Oligonucleotides and Analogs; Agrawal, ed.; Humana Press:Totowa, 1993, Vol. 20, 33-61; and the H-phosphonate method, as describedby Froehler in Methods in Molecular Biology: Protocols forOligonucleotides and Analogs; Agrawal, ed.; Humana Press: Totowa, 1993,Vol. 20, 63-80. Of these three methods, the phosphoramidite method hasbecome a de facto standard in the industry.

A typical oligonucleotide synthesis using phosphoramidite chemistry(i.e. the amidite methodology) is set forth below. First, a primersupport is provided in a standard synthesizer column. The primer supportis typically a solid support (supt) having a linker (link) covalentlybonded thereto. It is common to purchase the primer support with a first5′-protected nucleoside bonded thereto.

Primer support: bg is a 5′-blocking group, Bx is a nucleobase, R₂ is H,OH, OH protected with a removable protecting group, or a 2′-substituent,such as 2′-deoxy-2′-methoxyethoxy (2′-O-MOE), and link is the covalentlinking group, which joins the nucleoside to the support, supt.

-   -   (A) The 5′-blocking group bg (e.g. 4,4′-dimethoxytrityl) is        first removed (e.g. by exposing the 5′-blocked primer-support        bound nucleoside to an acid), thereby producing a support-bound        nucleoside of the formula:        Activated primer support: wherein supt is the solid support,        link is the linking group, Bx is a nucleobase, R₂, is H, OH, OH        protected with a removable protecting group, or a        2′-substituent.    -   (B) The column is then washed with acetonitrile, which acts to        both “push” the reagent (acid) onto the column, and to wash        unreacted reagent and the removed 5′-blocking group (e.g. trityl        alcohol) from the column.    -   (C) The primer support is then reacted with a phosphitylation        reagent (amidite), which is dissolved in acetonitrile, the        amidite having the formula:        wherein bg is a 5′-blocking group, lg is a leaving group, G is O        or S, pg is a phosphorus protecting group, and R_(2′) and Bx        have, independent of the analogous variables on the primer        support, the same definitions as previously defined.

The product of this reaction is the support-bound phosphite dimer:

Support-bound wherein each of the variables bg, pg, G, R_(2′) and Bx isindependently defined above, link is the linker and supt is the support,as defined above.

-   -   (D) The support-bound dimer is then typically washed with        acetonitrile.    -   (E) A capping reagent in acetonitrile is then added to the        column, thereby capping unreacted nucleoside.    -   (F) The column is then washed again with acetonitrile.    -   (G) The support-bound dimer is then typically reacted with an        oxidizing agent, such as a thiolating agent (e.g. phenylacetyl        disulfide), in acetonitrile, to form a support-bound phosphate        triester:    -   wherein G′ is O or S and the other variables are defined herein.    -   (H) The support-bound phosphate triester is then typically        washed with acetonitrile.

Steps (A)-(F) are then repeated, if necessary, a sufficient number oftimes to prepare a support-bound, blocked oligonucleotide having theformula:

wherein n is a positive integer (typically about 7 to about 79).

The phosphorus protecting groups pg are then typically removed from theoligomer to produce a support-bound oligomer having the formula:

which, after washing with a suitable wash solvent, such as acetonitrile,is typically cleaved from the solid support, purified, 5′-deblocked, andfurther processed to produce an oligomer of the formula:

The person having skill in the art will recognize that G′H bound to aP(V) phosphorus is generally is ionized at physiologic pH, and thattherefore, wherever G′H appears in the formulae above, or hereafter, G′⁻is synonymous therewith (the O⁻ or S⁻ being countered by a suitablecation, such as Na⁺).

A typical blocking group for 5′-protection of nucleotides is thedimethoxytrityl group (DMT). The DMT group is acid labile, and may beremoved with relatively weak acid, such as dichloroacetic acid. It isimportant that the oligonucleotide be produced in both good yield andexcellent purity. Yield is commonly expressed in terms of couplingefficiency, which is a measure of the degree to which each successivemonomer is coupled to the extant oligonucleotide. Coupling efficiency isaffected by a number of factors, including the choice of nucleosidemonomers, solvents, temperature, reagents, etc.

Purity is affected by a number of factors, including incomplete coupling(which produces so-called short-mers), as well as the introduction ofimpurities by reagents, solvents, etc.

It is a goal of oligonucleotide synthesis to produce large quantities ofoligonucleotides in excellent yield and purity. Despite advances in theart of oligonucleotide synthesis, there is still a need for syntheticmethods the produce oligonucleotides of improved purity.

SUMMARY OF THE INVENTION

The foregoing and further needs are met by embodiments of the presentinvention, which provide a process of oligonucleotide synthesiscomprising a deblocking step, wherein said deblocking step is carriedout in the substantial absence of chloral (trichloroacetaldehyde),chloral hydrate, and other derivatives thereof.

The foregoing and further needs are further met by embodiments of theinvention, which provide a process of oligonucleotide synthesis,comprising a dichloroacetic acid detritylation step, wherein saiddetritylation step is carried out in the substantial absence of chloral,its hydrates and other derivatives thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides improved methods of synthesizingoligonucleotides. In particular, the present invention provides improvedmethods of deblocking a protected group of a nucleoside duringoligonucleotide synthesis. The improved methods of the present inventioncomprise deblocking the nucleoside in the substantial absence of chloral(Cl₃CCHO), chloral hydrate (Cl₃CCH(OH)₂), and other derivatives ofchloral. The present invention leads to oligonucleotides having enhancedpurity as compared to oligonucleotides produced by previously knownmethodologies. The present invention furthermore leads tooligonucleotides in enhanced yields as compared to prior art methods.

The inventors have discovered that during normal oligonucleotidesynthesis a significant impurity arises out of the coupling of chloralor its derivatives to the nascent oligonucleotide during deblocking of5′-OH groups. Such deblocking is generally referred to as detritylation,because the trityl group, or DMT, is the most commonly used group forprotecting the 5′-OH of the nucleoside during coupling of the nucleosideto the oligonucleotide. The most commonly used reagent for detritylationis dichloroacetic acid (DCA).

The inventors have discovered that even very small amounts of chloralimpurity in DCA can lead to significant impurities arising from couplingof chloral to or within the oligonucleotide chain. In this context, theterm “chloral impurity” is intended to encompass chloral, chloralhydrate, other chloral derivatives present in DCA, and/or mixturesthereof. In contrast, when DCA that is substantially free of chloralimpurity is used as a detritylating reagent, the chloral adducts can besignificantly reduced or eliminated.

The present invention therefore contemplates deblocking a blockedoligonucleotide in the substantial absence of chloral, its hydrates andother derivatives thereof. In particular the present inventioncontemplates detritylation in the substantial absence of chloral, itshydrates and other derivatives thereof. More particularly, the presentinvention provides for detritylation in the presence of a dichloroaceticacid solution in the substantial absence of chloral impurity.

The present invention also contemplates deblocking reagents that aresubstantially free of chloral impurity. In particular, the presentinvention contemplates detritylating reagents that are substantiallyfree of chloral impurity. More particularly, the present inventionprovides dichloroacetic acid that is free of chloral impurity.

The present invention also contemplates oligonucleotides that aresubstantially free of chloral adducts, as described herein.

Common oligonucleotide synthesis is carried out by the phosphoramiditeprocess taught by Caruthers et al. (U.S. Pat. Nos. 4,458,066, 4,500,707,5,132,418, 4,415,732, 4,668,777 and 4,973,679) and Köster et al. (seee.g. U.S. Reissue 34,069). The phosphoramidite process generallyincludes a deblocking step for each cycle of chain extension, in whichthe 5′-OH is deblocked. In commercial production, the most commonly usedblocking group for the 5′-OH group is the 4,4′-dimethoxytriphenylmethyl(DMT) group, which is generally removed by treating the growingoligonucleotide with an acid, e.g. dichloroacetic acid.

The present inventors are the first to have recognized that thedeblocking step gives rise to certain adducts that are difficult toremove from the final oligomer product. In particular, the presentinventors have determined that the adducts of chloral, chloral hydrateor other chloral derivatives are produced during the detritylation stepwhen chloral impurity is present in the acid used to remove the DMTgroup from the oligonucleotide.

The physical properties of the chloral adducts are similar to those ofthe desired oligonucleotide products, which makes separation of chloraladducts from the desired oligonucleotide difficult.

The present inventors have surprisingly discovered that a very smallamount of chloral hydrate can have a significant impact on the purityand yield of the desired oligonucleotide product. In fact,concentrations of as little as 0.03 wt. % chloral hydrate (based onweight of 3% v/v DCA solution in toluene) can lead to significantchloral hydrate adducts in the oligonucleotide product.

The inventors have prepared dichloroacetic acid (DCA) that is lower inchloral impurity than commercially available DCA. Reduced chloralimpurity DCA was prepared by vacuum distillation. DCA fractionscontaining chloral impurity below the limit of detection were used inoligonucleotide synthesis. The used of such reduced chloral impurity DCAresulted in oligonucleotide product of significantly improved purity.

Other art recognized methods may be used to prepare reduced chloralimpurity DCA, however vacuum distillation is preferred for itsscalability. As it has been found that as little as 0.03 wt. % ofchloral hydrate in DCA can lead to significant occurrence of chloraladducts, it is desirable to use DCA that contains significantly lessthan 0.03 wt. % chloral hydrate. In certain embodiments according to thepresent invention, DCA containing chloral impurity below the limit ofdetection are used in the deblocking (e.g. detritylating) step ofoligonucleotide synthesis.

The present invention provides excellent purity and coupling efficiencyof oligonucleotide produced in oligonucleotide synthesis. While theinvention has been described with reference to certain preferredembodiments, it is to be understood that other embodiments are possiblewithin the scope of the present invention.

Oligonucleotides

The basic subunit of an oligonucleotide, such as RNA or DNA is depictedbelow.

In an oligonucleotide, Bx serves as the Binding Member, as describedabove, the phosphate moiety [P(=G′)(G″H)OH] serves as the LinkingMember, and the residue, referred to as the sugar backbone, is theBackbone Member. The phosphate member forms covalent bonds bycondensation with the 5′-OH of an adjacent subunit, thereby forming aphosphate diester bond. Where each of G′ and G″ is O, this is called aphosphodiester bond; where one of G′ or G″ is S and the other is O, thisis called a phosphorothioate bond, and where both G′ and G″ are S, thisis called a phosphorodithioate bond.

One skilled in the art will recognize that in naturally occurringnucleotides, R_(2′) is H for DNA (deoxyribonucleic acid) and OH for RNA(ribonucleic acid), each of G′ and G″ is O and Bx is one of thefollowing structures:

, wherein G, C, A, U and T are guanine, cytosine, adenine, thymine anduracil, respectively.

In the above formula, G′ and G″ may be O or S, and R_(2′) may be H, OHor some other value.

In naturally occurring RNA, the binding member is a nucleosidic baseselected from G, C, A and U, and the backbone comprises a sugar residue(ribosyl, i.e. R_(2′) is OH) and a phosphate (G′=G″═O). The ribosylsugar residue is the backbone member, while the phosphate joins adjacentmonomers through the 5′- and 3′-oxygen atoms on the ribosyl ring. Thesugar is covalently bound to the nucleosidic base (base) at the1′-position, the -β-D configuration predominating.

Naturally occurring DNA is analogous to RNA, except that the sugar is a2′-deoxyribosyl (R_(2′) is H).

Generally oligonucleotides according to the present invention includenaturally occurring and non-naturally occurring oligonucleotides. Ingeneral, oligonucleotides according to the present invention includecompounds of the formula (1):

wherein each Bx is a nucleobase as defined herein, each q is 0 or 1,each of R_(2′) is H, OH, reversibly-protected OH or a substituent ortogether with R_(4′) forms a bridge; R_(3′) is H or a substituent;R_(4′) is H, a substituent or together with R_(2′) or R_(5′) forms abridge; R_(5′) is H, a substituent or together with R_(4′) forms abridge, and each squiggly bond (

) indicates that the bond may be in the up or down configuration.

The naturally occurring oligonucleotides are those in which each of Bxis selected from the group consisting of G, C, A, U (for RNA) and T(DNA), each of G′ and G″ is O, each R_(3′), each R_(4′), each R_(5′) isH, each q is 1 and n is an integer, and the sugar oxygens are in theribosyl configuration. Conversely, non-naturally occurringoligonucleotides include those in which at least one of the followingconditions applies: At least one Bx is a nucleobase other than a memberselected from the groups consisting of G, C, A, U (for RNA) and T (DNA),at least one of G′ and G″ is other than O, at least one R_(3′), R_(4′),or R_(5′) is other than H, at least at least one q is 0, or at least oneof the sugar oxygens is in other than the ribosyl configuration. As usedherein, the term “oligonucleotide” encompasses both naturally occurringoligonucleotides and non-naturally occurring oligonucleotides, ormixtures thereof. In specific embodiments of the present invention, theterm oligonucleotide refers to a non-naturally occurring oligonucleotidehaving both naturally-occurring and non-naturally-occurring nucleotidesubunits. In specific embodiments of the invention, one or morenucleobases, sugar backbones and/or phosphate linking members arenon-naturally-occurring. These features will be described in greaterdetail below.

Sugar Backbone

In general, the sugar backbone has the structure:

wherein each Bx is a nucleobase as defined herein, q is 0 or 1, each ofR_(2′) is H, OH, reversibly-protected OH or a substituent or togetherwith R_(4′) forms a bridge; R_(3′) is H or a substituent; R_(4′) is H, asubstituent or together with R_(2′) or R_(5′) forms a bridge; R_(5′) isH, a substituent or together with R_(4′) forms a bridge. The dashes (

) indicate the positions at which the sugar moiety is bound to aphosphate linker to form a nucleotide bond.

The person skilled in the art will recognize that when R_(2′) is in thedown configuration and q′ is 1, the ring is a ribosyl ring, whereas whenR_(2′) is in the up configuration and q′ is 1, the ring is an arabinosylring. Likewise, when q′ is 0 and R₂′ is in the down configuration, thering is an erythrosyl ring. When R₂′ and R₄′ are joined to form abridge, the ring is called a locked nucleic acid (LNA), as described ingreater detail herein. In some embodiments, the bridge formed by R₂′ andR₄′ is R₂′—O—(CH₂)_(r)—R₄′ (wherein r is 1 or 2) or R₂′-CH₂—O—CH₂-R₄′(the use of R₂′ and R₄′ in the sub-formulae indicating the points ofattachment.) LNA may be present in either α-L- or β-D-conformation. SeeVester et al., “LNAzymes: Incorporation of LNA-Type Monomers intoDNAzymes Markedly Increases RNA Cleavage,” Journal of the AmericanChemical Society, 2002, 124, 13682-3. Each of these analogs possesses anumber of useful characteristics, including resistance to exonucleaseactivity, induction if endonuclease activity (e.g. by RNAse H, the RISCcomplex, etc.) and modulation of hybridization.

When R_(4′) and R_(5′) form a bridge, they may form, along with thesugar ring to which they are attached, a tricyclic ring. Tricyclicnucleosides of the structure:

are described by Rennenberg et al. in Nucleic Acids Research, 30(13),2751-7 (2002). One skilled in the art will recognize that the analogousphosphorothioates, and 2′-substituted tricyclic deoxynucleosides may beprepared by methods analogous to those taught by Rennenberg et al., asmodified by the teaching herein. In particular, the phosphorothioatesmay be prepared by substituting a sulfurizing oxidant (a.k.a. a sulfurtransfer reagent, such a phenyl acetyl disulfide) for the oxidizingagent taught by Rennenberg et al. The 2′-substituted tricyclicdeoxynucleosides may be prepared from the analogous 2′-substituteddeoxynucleosides, using a 2′-OH protecting group in the case ofribonucleic acid.

Certain oligonucleotides that utilized arabino-pentofuranosylnucleotides as building blocks have been described. Damha et. al.,J.A.C.S., 1998, 120, 12976-12977; and Damha et. al., Bioconjugate Chem.,1999, 10, 299-305.

Suitable 2′-substituents corresponding to R_(2′) include: F, O-alkyl(e.g. O-methyl), S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl;O-alkynyl, S-alkynyl, N-alkynyl; O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl or alkynyl, respectively. Particularly preferredare O[(CH₂)_(g)O]_(h)CH₃, O(CH₂)_(g)OCH₃, O(CH₂)_(g)NH₂, O(CH₂)_(g)CH₃,O(CH₂)_(g)ONH₂, and O(CH₂)_(g)ON[(CH₂)_(g)CH₃]₂, where g and h are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred 2′-modification is 2′-deoxy-2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE ribosyl)(Martin et al., Helv. Chim. Acta, 1995, 78, 486-504). Other preferredmodifications include 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (alsoknown in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE),i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.

Further representative substituent groups include groups of formulaI_(a) or II_(a):

wherein:

-   -   R_(b) is O, S or NH;    -   R_(d) is a single bond, O or C(═O);    -   R_(e) is C₁-C₁₀ alkyl, N(R_(k))(R_(m)), N(R_(k))(R_(n)),        N═C(R_(p))(R_(q)), N═C(R_(p))(R_(r)) or has formula III_(a);    -   each R_(c), R_(q), R_(r), R_(s), R_(t), R_(u) and R_(v) is,        independently, hydrogen, C(O)R_(w), substituted or unsubstituted        C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl,        substituted or unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl,        arylsulfonyl, a chemical functional group or a conjugate group,        wherein the substituent groups are selected from hydroxyl,        amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,        thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;    -   or optionally, R_(u) and R_(v), together form a phthalimido        moiety with the nitrogen atom to which they are attached;    -   each R_(w) is, independently, substituted or unsubstituted        C₁-C₁₀ alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy,        t-butoxy, allyloxy, 9-fluorenylmethoxy,        2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy,        butyryl, iso-butyryl, phenyl or aryl;    -   R_(k) is hydrogen, a nitrogen protecting group or —R_(x)-R_(y);    -   R_(p) is hydrogen, a nitrogen protecting group or —R_(x)-R_(y);    -   R_(x) is a bond or a linking moiety;    -   R_(y) is a chemical functional group, a conjugate group or a        solid support medium;    -   each R_(m) and R_(n) is, independently, H, a nitrogen protecting        group, substituted or unsubstituted C₁-C₁₀ alkyl, substituted or        unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted        C₂-C₁₀ alkynyl, wherein the substituent groups are selected from        hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,        thioalkoxy, halogen, alkyl, aryl, alkenyl, alkynyl; NH₃ ⁺,        N(R_(u))(R_(v)), guanidino and acyl where said acyl is an acid        amide or an ester;    -   or R_(m) and R_(n), together, are a nitrogen protecting group,        are joined in a ring structure that optionally includes an        additional heteroatom selected from N and O or are a chemical        functional group;    -   R_(i) is OR_(z), SR_(z), or N(R_(z))₂;    -   each R_(z) is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,        C(═NH)N(H)R_(u), C(═O)N(H)R_(u) or OC(═O)N(H)R_(u);    -   R_(f), R_(g) and R_(h) comprise a ring system having from about        4 to about 7 carbon atoms or having from about 3 to about 6        carbon atoms and 1 or 2 heteroatoms wherein said heteroatoms are        selected from oxygen, nitrogen and sulfur and wherein said ring        system is aliphatic, unsaturated aliphatic, aromatic, or        saturated or unsaturated heterocyclic;    -   R_(j) is alkyl or haloalkyl having 1 to about 10 carbon atoms,        alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to        about 10 carbon atoms, aryl having 6 to about 14 carbon atoms,        N(R_(k))(R_(m))OR_(k), halo, SR_(k) or CN;    -   ma is 1 to about 10;    -   mb is, independently, 0 or 1;    -   mc is 0 or an integer from 1 to 10;    -   md is an integer from 1 to 10;    -   me is from 0, 1 or 2; and    -   provided that when mc is 0, md is greater than 1.

Representative substituents groups of Formula I are disclosed in U.S.patent application Ser. No. 09/130,973, filed Aug. 7, 1998, entitled“Capped 2′-Oxyethoxy Oligonucleotides.” Representative cyclicsubstituent groups of Formula II are disclosed in U.S. patentapplication Ser. No. 09/123,108, filed Jul. 27, 1998, entitled “RNATargeted 2′-Modified Oligonucleotides that are ConformationallyPreorganized.”

Particularly preferred sugar substituent groups includeO[(CH₂)_(g)O]_(h)CH₃, O(CH₂)_(g)OCH₃, O(CH₂)_(g)NH₂, O(CH₂)_(g)CH₃,O(CH₂)_(g)ONH₂, and O(CH₂)_(g)ON[(CH₂)_(g)CH₃)]₂, where g and h are from1 to about 10.

Some preferred oligomeric compounds of the invention contain at leastone nucleoside having one of the following substituent groups: C₁ to C₁₀lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl orO-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂,NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,poly-alkylamino, substituted silyl, an RNA cleaving group, a reportergroup, an intercalator, a group for improving the pharmacokineticproperties of an oligomeric compound, or a group for improving thepharmacodynamic properties of an oligomeric compound, and othersubstituents having similar properties. A preferred modificationincludes 2′-methoxyethoxy [2′-O-CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE] (Martin et al., Helv. Chim. Acta, 1995,78, 486), i.e., an alkoxyalkoxy group. A further preferred modificationis 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also knownas 2′-DMAOE. Representative aminooxy substituent groups are described inco-owned U.S. patent application Ser. No. 09/344,260, filed Jun. 25,1999, entitled “Aminooxy-Functionalized Oligomers”; and U.S. patentapplication Ser. No. 09/370,541, filed Aug. 9, 1999, entitled“Aminooxy-Functionalized Oligomers and Methods for Making Same.”

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on nucleosides andoligomers, particularly the 3′ position of the sugar on the 3′ terminalnucleoside or at a 3′-position of a nucleoside that has a linkage fromthe 2′-position such as a 2′-5′ linked oligomer and at the 5′ positionof a 5′ terminal nucleoside. Oligomers may also have sugar mimetics suchas cyclobutyl moieties in place of the pentofuranosyl sugar.Representative United States patents that teach the preparation of suchmodified sugars structures include, but are not limited to, U.S. Pat.Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265; 5,658,873;5,670,633; and 5,700,920, and commonly owned U.S. patent applicationSer. No. 08/468,037, filed on Jun. 5, 1995.

Representative guanidino substituent groups that are shown in formulaIII and IV are disclosed in co-owned U.S. patent application Ser. No.09/349,040, entitled “Functionalized Oligomers”, filed Jul. 7, 1999.

Representative acetamido substituent groups are disclosed in U.S. Pat.No. 6,147,200. Representative dimethylaminoethyloxyethyl substituentgroups are disclosed in International Patent Application PCT/US99/17895,entitled “2′-O-Dimethylaminoethyloxyethyl-Modified Oligonucleotides”,filed Aug. 6, 1999. For those nucleosides that include a pentofuranosylsugar, the phosphate group can be linked to either the 2′, 3′ or 5′hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphategroups covalently link adjacent nucleosides to one another to form alinear polymeric compound. The respective ends of this linear polymericstructure can be joined to form a circular structure by hybridization orby formation of a covalent bond, however, open linear structures aregenerally preferred. Within the oligonucleotide structure, the phosphategroups are commonly referred to as forming the internucleoside linkagesof the oligonucleotide. The normal internucleoside linkage of RNA andDNA is a 3′ to 5′ phosphodiester linkage.

While the present invention may be adapted to produce oligonucleotidesfor any desired end use (e.g. as probes for us in the polymerase chainreaction), one preferred use of the oligonucleotides is in antisensetherapeutics. One mode of action that is often employed in antisensetherapeutics is the so-called RNAse H mechanism, whereby a strand of DNAis introduced into a cell, where the DNA hybridizes to a strand of RNA.The DNA-RNA hybrid is recognized by an endonuclease, RNAse H, whichcleaves the RNA strand. In normal cases, the RNA strand is messenger RNA(mRNA), which, after it has been cleaved, cannot be translated into thecorresponding peptide or protein sequence in the ribosomes. In this way,DNA may be employed as an agent for modulating the expression of certaingenes.

It has been found that by incorporating short stretches of DNA into anoligonucleotide, the RNAse H mechanism can be effectively used tomodulate expression of target peptides or proteins. In some embodimentsof the invention, an oligonucleotide incorporating a stretch of DNA anda stretch of RNA or 2′-modified RNA can be used to effectively modulategene expression. In preferred embodiments, the oligonucleotide comprisesa stretch of DNA flanked by two stretches of 2′-modified RNA. Preferred2′-modifications include 2′-O-methyl and 2′-O-methoxyethyl as describedherein.

The ribosyl sugar moiety has also been extensively studied to evaluatethe effect its modification has on the properties of oligonucleotidesrelative to unmodified oligonucleotides. The 2′-position of the sugarmoiety is one of the most studied sites for modification. Certain2′-substituent groups have been shown to increase the lipohpilicity andenhance properties such as binding affinity to target RNA, chemicalstability and nuclease resistance of oligonucleotides. Many of themodifications at the 2′-position that show enhanced binding affinityalso force the sugar ring into the C₃-endo conformation.

RNA exists in what has been termed “A Form” geometry while DNA exists in“B Form” geometry. In general, RNA:RNA duplexes are more stable, or havehigher melting temperatures (Tm) than DNA:DNA duplexes (Sanger et al.,Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New York,N.Y.; Lesnik et al., Biochemistry, 1995, 34, 10807-10815; Conte et al.,Nucleic Acids Res., 1997, 25, 2627-2634). The increased stability of RNAhas been attributed to several structural features, most notably theimproved base stacking interactions that result from an A-form geometry(Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The presenceof the 2′ hydroxyl in RNA biases the sugar toward a C3′ endo pucker,i.e., also designated as Northern pucker, which causes the duplex tofavor the A-form geometry. On the other hand, deoxy nucleic acids prefera C2′ endo sugar pucker, i.e., also known as Southern pucker, which isthought to impart a less stable B-form geometry (Sanger, W. (1984)Principles of Nucleic Acid Structure, Springer-Verlag, New York, N.Y.).In addition, the 2′ hydroxyl groups of RNA can form a network of watermediated hydrogen bonds that help stabilize the RNA duplex (Egli et al.,Biochemistry, 1996, 35, 8489-8494).

DNA:RNA hybrid duplexes, however, are usually less stable than pureRNA:RNA duplexes, and depending on their sequence may be either more orless stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res.,1993, 21, 2051-2056). The structure of a hybrid duplex is intermediatebetween A- and B-form geometries, which may result in poor stackinginteractions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306;Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al.,Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996,264, 521-533). The stability of a DNA:RNA hybrid is central to antisensetherapies as the mechanism requires the binding of a modified DNA strandto a mRNA strand. To effectively inhibit the mRNA, the antisense DNAshould have a very high binding affinity with the mRNA. Otherwise thedesired interaction between the DNA and target mRNA strand will occurinfrequently, thereby decreasing the efficacy of the antisenseoligonucleotide.

Various synthetic modifications have been proposed to increase nucleaseresistance, or to enhance the affinity of the antisense strand for itstarget mRNA (Crooke et al., Med. Res. Rev., 1996, 16, 319-344; DeMesmaeker et al., Acc. Chem. Res., 1995, 28, 366-374). A variety ofmodified phosphorus-containing linkages have been studied asreplacements for the natural, readily cleaved phosphodiester linkage inoligonucleotides. In general, most of them, such as thephosphorothioate, phosphoramidates, phosphonates and phosphorodithioatesall result in oligonucleotides with reduced binding to complementarytargets and decreased hybrid stability.

One synthetic 2′-modification that imparts increased nuclease resistanceand a very high binding affinity to nucleotides is the 2′-methoxyethoxy(MOE, 2′-OCH₂CH₂OCH₃) side chain (Baker et al., J. Biol. Chem., 1997,272, 11944-12000; Freier et al., Nucleic Acids Res., 1997, 25,4429-4443). One of the immediate advantages of the MOE substitution isthe improvement in binding affinity, which is greater than many similar2′ modifications such as O-methyl, O-propyl, and O-aminopropyl (Freierand Altmann, Nucleic Acids Research, (1997) 25:4429-4443).2′-O-methoxyethyl-substituted oligonucleotides also have been shown tobe antisense inhibitors of gene expression with promising features forin vivo use (Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Altmann etal., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans.,1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997,16, 917-926). Relative to DNA, they display improved RNA affinity andhigher nuclease resistance. Chimeric oligonucleotides with2′-O-methoxyethyl-ribonucleoside wings and a centralDNA-phosphorothioate window also have been shown to effectively reducethe growth of tumors in animal models at low doses. MOE substitutedoligonucleotides have shown outstanding promise as antisense agents inseveral disease states. One such MOE substituted oligonucleotide ispresently being investigated in clinical trials for the treatment of CMVretinitis.

LNAs (oligonucleotides wherein the 2′ and 4′ positions are connected bya bridge) also form duplexes with complementary DNA, RNA or LNA withhigh thermal affinities. Circular dichroism (CD) spectra show thatduplexes involving fully modified LNA (esp. LNA:RNA) structurallyresemble an A-form RNA:RNA duplex. Nuclear magnetic resonance (NMR)examination of an LNA:DNA duplex confirmed the 3′-endo conformation ofan LNA monomer. Recognition of double-stranded DNA has also beendemonstrated suggesting strand invasion by LNA. Studies of mismatchedsequences show that LNAs obey the Watsor-Crick base pairing rules withgenerally improved selectivity compared to the corresponding unmodifiedreference strands. LNAs may be in either the α-L- or theβ-D-conformation. Vester et al., J.A.C.S, 124 (2002) 13682-13683.

LNAs in which the 2′-hydroxyl group is linked to the 4′ carbon atom ofthe sugar ring thereby forming a 2′-C,4′-C-oxymethylene linkage therebyforming a bicyclic sugar moiety. The linkage is preferably an alkylene((—CH₂—)_(n)) group bridging the 2′ oxygen atom and the 4′ carbon atomwherein n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456). LNAand LNA analogs display very high duplex thermal stabilities withcomplementary DNA and RNA (Tm=+3 to +10 C), stability towards3′-exonucleolytic degradation and good solubility properties. Otherpreferred bridge groups include the 2′-CH₂OCH₂-4′ bridge.

Nucleobases

The nucleobases Bx (also referred to in the art as nucleic acid bases orsimply as bases) may be naturally-occurring G, C, A, U or T, or may beselected from a wide range of non-naturally occurring bases as describedherein. The two most common classes of nucleobases are purines andpyrimidines. The naturally-occurring purine bases are guanine (G) andadenine (A), which are linked to the sugar through the 9-N nitrogen inthe β-anomeric position on the sugar ring. The naturally-occurringpyrimidine bases are uracil (U), thymine (T) and cytidine (C), which arelinked to the sugar through the 1-N nitrogen. In double stranded DNA(dsDNA), Watson-Crick base pairing occurs between G and C, and between Aand T, whereas in double stranded RNA (dsRNA), Watson-Crick base pairingoccurs between G and C, and between A and U. The Watson-Crick base pairsfor DNA and RNA are shown below.

Analogous base pairing is generally observed in RNA-DNA hybrids, as wellas in hybrids between naturally-occurring RNA or DNA and syntheticoligonucleotides comprising non naturally occurring monomeric subunits.

In synthetic oligonucleotides according to the invention, such asantisense therapeutics and diagnostics, one or more of thenaturally-occurring nucleobases may be replaced by an analogous bindingmember (nucleobase analog). Thus, the term “nucleobase” encompasses bothnaturally-occurring and non-naturally-occurring nucleobases. The term“nucleobase analog” (also referred to herein is a nucleobase mimetic ora nucleic acid base mimetic) refers to non-naturally-occurringnucleobases, and means a residue that functions like a nucleobase byproviding sequence specific binding to a heterocyclic residue on acomplementary oligomer. In some embodiments according to the invention,a nucleobase analog is a residue that is capable of establishing one ormore non-covalent bonds with a nucleobase on a separate oligonucleotidestrand. Non-covalent bonds are hydrogen bonds, ionic bonds and polarinteractions. (Additional interactions with non-complementarynucleobases are also possible, such as base-stacking interactions). Insome embodiments of the invention, non-covalent bonds are formed byhydrogen bonding between nucleobase ring constituents and/or exocyclicsubstituents, and may be analogous to Watson-Crick bonding, Hoogsteenbonding, some combination thereof, or some other regime as describedherein or as known in the art.

As used herein, “unmodified” or “natural” nucleobases mean the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases (nucleobase analogs)include other synthetic and natural nucleobases such as 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynylderivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine,5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine,7-propynyl-7-deaza-8-azaguanine, 7-propynyl-7-deaza-8-azaadenine.Further modified nucleobases include tricyclic pyrimidines such asphenoxazine cytidine(1H-pyrimido[5,4b][1,4]benzoxazin-2(3H)-one),phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one),G-clamps such as a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993.

Certain of these nucleobases are particularly useful for increasing thebinding affinity of the oligomeric compounds of the invention. Theseinclude 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, and U.S. Pat. No. 5,750,692.

In general, the term “base” includes the term nucleobase as describedabove. The term “base” means a binding member, as described hereinabove.While nucleobases are generally heterocyclic moieties, the term “base”as used herein with means any moiety or residue capable of participatingin specific binding to a naturally-occurring nucleobase.

In some embodiments of the present invention oligomeric compounds areprepared having polycyclic heterocyclic compounds in place of one ormore heterocyclic base moieties. A number of tricyclic heterocycliccompounds have been previously reported. These compounds are routinelyused in antisense applications to increase the binding properties of themodified strand to a target strand. The most studied modificationsselectively bind to guanosines. Hence they have been termed G-clamps orcytidine analogs. Many of these polycyclic heterocyclic compounds havethe general formula:

Representative cytosine analogs that make 3 hydrogen bonds with aguanosine in a second strand include 1,3-diazaphenoxazine-2-one (R₁₀═O,R₁₁-R₁₄═H) [Kurchavov, et al., Nucleosides and Nucleotides, 1997, 16,1837-1846], 1,3-diazaphenothiazine-2-one (R₁₀═S, R₁₁-R₁₄═H), [Lin,K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117,3873-3874] and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (R₁₀═O,R₁₁-R₁₄═F) [Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998,39, 8385-8388]. Incorporated into oligonucleotides these basemodifications were shown to hybridize with complementary guanine and thelatter was also shown to hybridize with adenine and to enhance helicalthermal stability by extended stacking interactions (also see U.S.patent application entitled “Modified Peptide Nucleic Acids” filed May24, 2002, Ser. No. 10/155,920; and U.S. patent application entitled“Nuclease Resistant Chimeric Oligonucleotides” filed May 24, 2002, Ser.No. 10/013,295). R₁₅ in these structures is typically O but can also beS.

Further helix-stabilizing properties have been observed when a cytosineanalog/substitute has an aminoethoxy moiety attached to the rigid1,3-diazaphenoxazine-2-one scaffold (R₁₀═O, R₁₁=—O—(CH₂)₂—NH₂, R₁₂₋₁₄═H)[Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532].Binding studies demonstrated that a single incorporation could enhancethe binding affinity of a model oligonucleotide to its complementarytarget DNA or RNA with aΔT_(m) of up to 18° relative to 5-methylcytosine (dC5^(me)), which is the highest known affinity enhancement fora single modification, yet. On the other hand, the gain in helicalstability does not compromise the specificity of the oligonucleotides.The T_(m) data indicate an even greater discrimination between theperfect match and mismatched sequences compared to dC5^(me). It wassuggested that the tethered amino group serves as an additional hydrogenbond donor to interact with the Hoogsteen face, namely the O6, of acomplementary guanine thereby forming 4 hydrogen bonds. This means thatthe increased affinity of G-clamp is mediated by the combination ofextended base stacking and additional specific hydrogen bonding.

Further tricyclic heterocyclic compounds and methods of using them thatare amenable to the present invention are disclosed in U.S. Pat. No.6,028,183, which issued on May 22, 2000, and U.S. Pat. No. 6,007,992,which issued on Dec. 28, 1999. Such compounds include those having theformula:

wherein R₁₁ includes (CH₃)₂N—(CH₂)₂—O—; H₂N—(CH₂)₃—;Ph-CH₂—O—C(═O)N(H)—(CH₂)₃—; H₂N—; Fluorenyl-CH₂—O—C(═O)—N(H)—(CH₂)₃—;Phthalimidyl-CH₂—O—C(═O)—N(H)—(CH₂)₃—; Ph-CH₂—O—C(═O)—N(H)—(CH₂)₂—O—;Ph-CH₂—O—C(═O)—N(H)—(CH₂)₃—O—; (CH₃)₂N—N(H)—(CH₂)₂—O—;Fluorenyl-CH₂—O—C(═O)—N(H)—(CH₂)₂—O—;Fluorenyl-CH₂—O—C(═O)—N(H)CH₂)₃—O—; H₂N—(CH₂)₂—O—CH₂—; N₃—(CH₂)₂—O—CH₂—;H₂N—(CH₂)₂—O—, and NH₂C(═NH)NH—.

Also disclosed are tricyclic heterocyclic compounds of the formula:

wherein

R_(10a) is O, S or N—CH₃;

R_(11a) is A(Z)_(x1), wherein A is a spacer and Z independently is alabel bonding group bonding group optionally bonded to a detectablelabel, but R_(11a) is not amine, protected amine, nitro or cyano;

X1 is 1, 2 or 3; and

R_(b) is independently —CH═, —N═, —C(C₁₋₈ alkyl)= or —C(halogen)=, butno adjacent R_(b) are both —N═, or two adjacent R_(b) are taken togetherto form a ring having the structure:

where R_(c) is independently —CH═, —N═, —C(C₁₋₈ alkyl)═ or —C(halogen)═,but no adjacent R_(b) are both —N═.

The enhanced binding affinity of the phenoxazine derivatives togetherwith their uncompromised sequence specificity makes them valuablenucleobase analogs for the development of more potent antisense-baseddrugs. In fact, promising data have been derived from in vitroexperiments demonstrating that heptanucleotides containing phenoxazinesubstitutions are capable to activate RNaseH, enhance cellular uptakeand exhibit an increased antisense activity [Lin, K.-Y.; Matteucci, M.J. Am. Chem. Soc. 1998, 120, 8531-8532]. The activity enhancement waseven more pronounced in case of G-clamp, as a single substitution wasshown to significantly improve the in vitro potency of a 20mer2′-deoxyphosphorothioate oligonucleotides [Flanagan, W. M.; Wolf, J. J.;Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci, M. Proc.Natl. Acad. Sci. USA, 1999, 96, 3513-3518]. Nevertheless, to optimizeoligonucleotide design and to better understand the impact of theseheterocyclic modifications on the biological activity, it is importantto evaluate their effect on the nuclease stability of the oligomers.

Further tricyclic and tetracyclic heteroaryl compounds amenable to thepresent invention include those having the formulas:

wherein R₁₄ is NO₂ or both R₁₄ and R₁₂ are independently —CH₃. Thesynthesis of these compounds is disclosed in U.S. Pat. No. 5,434,257,which issued on Jul. 18, 1995, U.S. Pat. No. 5,502,177, which issued onMar. 26, 1996, and U.S. Pat. No. 5,646,269, which issued on Jul. 8,1997.

Further tricyclic heterocyclic compounds amenable to the presentinvention also disclosed in the “257, 177 and 269” Patents include thosehaving the formula:

a and b are independently 0 or 1 with the total of a and b being 0 or 1;

A is N, C or CH;

X is S, O, C═O, NH or NCH₂, R⁶;

Y is C═O;

Z is taken together with A to form an aryl or heteroaryl ring structurecomprising 5 or 6 ring atoms wherein the heteroaryl ring comprises asingle O ring heteroatom, a single N ring heteroatom, a single S ringheteroatom, a single 0 and a single N ring heteroatom separated by acarbon atom, a single S and a single N ring heteroatom separated by a Catom, 2 N ring heteroatoms separated by a carbon atom, or 3 N ringheteroatoms at least 2 of which are separated by a carbon atom, andwherein the aryl or heteroaryl ring carbon atoms are unsubstituted withother than H or at least I non-bridging ring carbon atom is substitutedwith R²⁰ or ═O;

or Z is taken together with A to form an aryl ring structure comprising6 ring atoms wherein the aryl ring carbon atoms are unsubstituted withother than H or at least 1 non-bridging ring carbon atom is substitutedwith R⁶ or ═O;

R⁶ is independently H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, NO₂,N(R³)₂, CN or halo, or an R⁶ is taken together with an adjacent Z groupR⁶ to complete a phenyl ring;

R²⁰ is, independently, H, C₁₋₆ alkyl, C₂₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, NO₂, N(R²¹)₂, CN, or halo, or an R²⁰ is taken together with anadjacent R²⁰ to complete a ring containing 5 or 6 ring atoms, andtautomers, solvates and salts thereof;

R²¹ is, independently, H or a protecting group;

R³ is a protecting group or H; and tautomers, solvates and saltsthereof.

More specific examples included in the “257, 177 and 269” Patents arecompounds of the formula:

wherein each R₁₆, is, independently, selected from hydrogen and varioussubstituent groups.

Further polycyclic base moieties having the formula:

wherein:

A₆ is O or S;

A₇ is CH₂, N—CH₃, O or S;each A₈ and A₉ is hydrogen or one of A₈ and A₉ is hydrogen and the otherof A₈ and A₉ is selected from the group consisting of:

wherein:

-   -   G is —CN, —OA₁₀, —SA₁₀, —N(H)A₁₀, —ON(H)A₁₀ or —C(═NH)N(H)A₁₀;    -   Q₁ is H, —NHA₁₀, —C(═O)N(H)A₁₀, —C(═S)N(H)A₁₀ or —C(═NH)N(H)A₁₀;    -   each Q₂ is, independently, H or Pg;    -   A₁₀ is H, Pg, substituted or unsubstituted C₁-C₁₀ alkyl, acetyl,        benzyl, —(CH₂)_(p3)NH₂, —(CH₂)_(p3)N(H)Pg, a D or L α-amino        acid, or a peptide derived from D, L or racemic α-amino acids;    -   Pg is a nitrogen, oxygen or thiol protecting group;    -   each p1 is, independently, from 2 to about 6;    -   p2 is from 1 to about 3; and    -   p3 is from 1 to about 4;        are disclosed in U.S. patent application Ser. No. 09/996,292        filed Nov. 28, 2001.

In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable.

Phosphate Linkers

Oligonucleotides are generally those oligomers in which the monomericsubunits comprise linking members having pentavalent phosphorus as aconstituent part. Phosphate linkers include phosphodiester,phosphorothioate and phosphorodithioate linkers.

wherein the squiggles (

) indicate covalent bonds to backbone members, e.g. oxygen atoms onsugar backbone moieties, or other substituent on sugar analogs.

Oligonucleotides as defined herein generally include salts, solvates andtautomers of oligonucleotides. In general, many bases, especiallynucleobases, can form tautomeric structures that are included within thegeneral definitions of oligonucleotides according to the presentinvention. In addition, the phosphorothioate linker can form thefollowing tautomers:

and can likewise form the following salt structures:

wherein M⁺ is a suitable salt-forming cation, such as Na⁺, K⁺, ½Ca²⁺, ½Mg²⁺, ⅓ Al³⁺, NH₄ ⁺, H₃O⁺, etc. (The fractions indicate fractionalequivalents of the cationic species per phosphate diester linkage.)Phosphodiester and phosphorodithioate moieties can form analogous salts.

Naturally occurring nucleosides are linked to one another via aphosphodiester linker. Antisense compounds may be prepared usingphosphodiester linkers, which are generally suitable for diagnostic andother nuclease-free uses. However, antisense therapeutic compoundsadvantageously comprise at least one phosphorothioate linker, owing tothe latter's superior nuclease stability. Both phosphodiester andphosphorothioate diester linkers are generally referred to as phosphatediester linkers. When a plurality of nucleotides are linked bysuccessive phosphate diester linkers, the resulting oligomer is calledan oligonucleotide.

Manufacture of Oligonucleotides

As described above, the term “oligonucleotide” encompassesnaturally-occurring RNA and DNA as well as phosphate-linked oligomershaving a variety of sugar backbones and nucleobases. Oligonucleotideshave been made by the phosphate triester, H-phosphonate andphosphoramidite methods as described hereinabove. Of these threemethods, the phosphoramidite method has become the de facto standard foroligonucleotide synthesis, especially where one or more modificationsare made to the sugar backbone or nucleobases, or where exceptionalpurity, yield or scale are paramount. The phosphoramidite method(amidite method) is described hereinafter.

Amidite Method

Oligonucleotides according to embodiments of the present invention arerepresented by formula 1, above.

While the present invention is concerned primarily witholigonucleotides, some oligonucleotide mimetics may, with appropriatechanges to the starting materials, also be prepared by processesaccording to the present invention. Oligonucleotide mimetics includecompounds in which the oligonucleotide sugar has been replaced with aheterocyclic or carbocyclic ring structure. Such compounds are depictedin Formula 1a, below.

and tautomers, salts and solvates thereof, wherein G, G′, Bx, n, R₂′,R_(3′), R_(4′) and R_(5′) each have the meanings previously defined. Thegroups T′ and T″ are each H, or conjugate groups, such as protectinggroups and substituents. Each Q′ is independently O, S, NR′″, C(R′″)₂,or —CR′″═CR′″—, where each R′″ is H, alkyl, or where two R′″ groups areon the same or adjacent carbon atoms, they may form a carbocyclic orheterocyclic ring, wherein the ring contains one or two of N, O or S.Preferred values of R′″ are H and C₁-C₄ alkyl.

The foregoing oligonucleotides and oligonucleotide mimetics may bemanufactured by solid phase synthesis, e.g. by the amidite method.Equipment for such synthesis is sold by several vendors including, forexample, Applied Biosystems (Foster City, Calif.). Other means for suchsynthesis known in the art may additionally or alternatively beemployed. For example stirred bed reactors have been used.

Support bound oligonucleotide synthesis relies on sequential addition ofnucleotides to one end of a growing chain. Typically, a first nucleoside(having protecting groups on any exocyclic amine functionalitiespresent) is attached to an appropriate glass bead support and activatedphosphite compounds (typically nucleotide phosphoramidites, also bearingappropriate protecting groups) are added stepwise to elongate thegrowing oligonucleotide. Additional methods for solid-phase synthesismay be found in Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066;4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Pat. No.4,725,677 and Re. 34,069.

Examples of the synthesis of particular modified oligonucleotides may befound in the following U.S. patents or pending patent applications, eachof which is commonly assigned with this application: U.S. Pat. Nos.5,138,045 and 5,218,105, drawn to polyamine conjugated oligonucleotides;U.S. Pat. No. 5,212,295, drawn to monomers for the preparation ofoligonucleotides having chiral phosphorus linkages; U.S. Pat. Nos.5,378,825 and 5,541,307, drawn to oligonucleotides having modifiedbackbones; U.S. Pat. No. 5,386,023, drawn to backbone modifiedoligonucleotides and the preparation thereof through reductive coupling;U.S. Pat. No. 5,457,191, drawn to modified nucleobases based on the3-deazapurine ring system and methods of synthesis thereof; U.S. Pat.No. 5,459,255, drawn to modified nucleobases based on N-2 substitutedpurines; U.S. Pat. No. 5,521,302, drawn to processes for preparingoligonucleotides having chiral phosphorus linkages; U.S. Pat. No.5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746,drawn to oligonucleotides having β-lactam backbones; U.S. Pat. No.5,571,902, drawn to methods and materials for the synthesis ofoligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides havingalkylthio groups, wherein such groups may be used as linkers to othermoieties attached at any of a variety of positions of the nucleoside;U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides havingphosphorothioate linkages of high chiral purity; U.S. Pat. No.5,506,351, drawn to processes for the preparation of 2′-O-alkylguanosine and related compounds, including 2,6-diaminopurine compounds;U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotideshaving 3-deazapurines; U.S. Pat. No. 5,223,168, issued Jun. 29, 1993,and 5,608,046, both drawn to conjugated 4′-desmethyl nucleoside analogs;U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn to backbone modifiedoligonucleotide analogs; and U.S. patent application Ser. No.08/383,666, filed Feb. 3, 1995, and U.S. Pat. No. 5,459,255, drawn to,inter alia, methods of synthesizing 2′-fluoro-oligonucleotides.

The amidite method of oligonucleotide synthesis may be carried outgenerally in the following manner: Phosphoramidites are prepared byreacting a suitable nucleoside or modified nucleoside (formula 4) with aphosphorodiamidite (formula 5) to form a phosphoramidite (formula 6):

wherein each of the variables Q′, Bx, R_(2′), R_(3′), R_(4′), R_(5′),G′, and q′ is as previously defined. L is an amine leaving group; pg isa phosphorus protecting group; and T′″ is a hydroxyl protecting group,each as more specifically defined herein. In some embodiments of thepresent invention, in at least one cycle of the synthetic method, T′″ isDMT.

A support-bound nucleoside of Formula 7 is first deprotected at the5′-position (resulting in a free 5′-OH group). In some embodiments ofthe present invention, at least one of the 5-protecting groups (T′″) isDMT, and the deprotecting reagent is dichloroacetic acid (DCA). In morespecific embodiments of the present invention, a plurality of5′-deprotection steps are carried out in the presence of DCA. In certainembodiments of the present invention, each of the 5′-deprotection stepsis carried out in the presence of DCA. In this context, DCA issubstantially free of chloral, chloral hydrate, or other derivatives ofchloral. In some embodiments, the DCA is tested by HPLC or othersuitable method and contains no chloral, chloral hydrate or otherderivative of chloral above the limit of detection.

After 5′-deprotection, a first amidite (7) is coupled to a support-boundnucleoside to form a support-bound dimer of Formula 8, which is thenoxidized, and subjected to a capping step to form a support bound dimerof Formula 9.

The 5′-deprotection, coupling, oxidation and capping steps are thenrepeated n-2 times to form a support-bound oligomer of Formula 10.

This compound (10) is then cleaved from the solid support,5′-deprotected, if necessary, and purified to yield an oligomer ofFormula (1). The oligonucleotide may then be further derivatized,purified, precipitated, or otherwise treated, as described in moredetail herein. In select embodiments of the present invention, the finalprotecting group is left on the oligonucleotide (10, SS replaced by H),which is first subjected to high performance liquid chromatography(HPLC), before the final 5′-protecting group is removed. In specificembodiments of the present invention, the final 5′-protecting group isremoved by contacting the purified oligonucleotide with acetic acid. Inother embodiments the 5′-protecting group may be removed while theoligonucleotide is left on the solid support (SS). The deprotectedoligonucleotide (10, wherein T′″ is replaced by H) may then be removedfrom the column as described above and subjected to purification steps.In specific embodiments of the invention, a deprotected oligonucleotidemay be subjected to ion exchange chromatography, such as soft anionexchange (SAX) chromatography. Anion exchange chromatography may becarried out either directly after a deprotected oligonucleotide isremoved from the solid synthesis support, or after a 5′-protectedoligonucleotide has been purified by liquid chromatography and thendeprotected.

In each of the foregoing Formulae, SS represents a support bound to the3′-terminal nucleoside by a cleavable linker, each pg is a phosphorusprotecting group as defined herein, n is an integer, G and G′ areindependently O or S, and each Bx, R_(2′), R_(3′), R′₄, R_(5′), Q′, andq′ is independently as defined in Formula 3.

Amidites

Phosphoramidites (amidites) used in the synthesis of oligonucleotidesare available from a variety of commercial sources (included are: GlenResearch, Sterling, Va.; Amersham Pharmacia Biotech Inc., Piscataway, N.J.; Cruachem Inc., Aston, Pa.; Chemgenes Corporation, Waltham, Mass.;Proligo LLC, Boulder, Colo.; PE Biosystems, Foster City Calif.; BeckmanCoulter Inc., Fullerton, Calif.). These commercial sources sell highpurity phosphoramidites generally having a purity of better than 98%.Those not offering an across the board purity for all amidites sold willin most cases include an assay with each lot purchased giving at leastthe purity of the particular phosphoramidite purchased. Commerciallyavailable phosphoramidites are prepared for the most part for automatedDNA synthesis and as such are prepared for immediate use forsynthesizing desired sequences of oligonucleotides. Phosphoramidites maybe prepared by methods disclosed by e.g. Caruthers et al. (U.S. Pat.Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and5,132,418) and Koster et al. (U.S. RE 34,069).

Support Media

Oligonucleotides are generally prepared, as described above, on asupport medium (support), e.g. a solid support medium. In general afirst synthon (e.g. a monomer, such as a nucleoside) is first attachedto a support medium, and the oligonucleotide is then synthesized bysequentially coupling monomers to the support-bound synthon. Thisiterative elongation eventually results in a final oligomeric compoundor other polymer such as a polypeptide. Suitable support media can besoluble or insoluble, or may possess variable solubility in differentsolvents to allow the growing support bound polymer to be either in orout of solution as desired. Traditional support media such as solidsupports are for the most part insoluble and are routinely placed inreaction vessels while reagents and solvents react with and/or wash thegrowing chain until the oligomer has reached the target length, afterwhich it is cleaved from the support and, if necessary further worked upto produce the final polymeric compound. More recent approaches haveintroduced soluble supports including soluble polymer supports to allowprecipitating and dissolving the iteratively synthesized product atdesired points in the synthesis (Gravert et al., Chem. Rev., 1997, 97,489-510).

The term support media (support) is intended to include supports knownto the person skilled in the art to for the synthesis of oligomericcompounds and related compounds such as peptides. Some representativesupport media that are amenable to the methods of the present inventioninclude but are not limited to the following: controlled pore glass(CPG); oxalyl-controlled pore glass (see, e.g., Alul, et al., NucleicAcids Research 1991, 19, 1527); silica-containing particles, such asporous glass beads and silica gel such as that formed by the reaction oftrichloro-[3-(4-chloromethyl)phenyl]propylsilane and porous glass beads(see Parr and Grohmann, Angew. Chem. Internal. Ed. 1972, 11, 314, soldunder the trademark “PORASIL E” by Waters Associates, Framingham, Mass.,USA); the mono ester of 1,4-dihydroxymethylbenzene and silica (see Bayerand Jung, Tetrahedron Lett., 1970, 4503, sold under the trademark“BIOPAK” by Waters Associates); TENTAGEL (see, e.g., Wright, et al.,Tetrahedron Letters 1993, 34, 3373); cross-linked styrene/divinylbenzenecopolymer beaded matrix or POROS, a copolymer ofpolystyrene/divinylbenzene (available from Perceptive Biosystems);soluble support media, polyethylene glycol PEG's (see Bonora et al.,Organic Process Research & Development, 2000, 4, 225-231).

Further support media amenable to the present invention include withoutlimitation PEPS support a polyethylene (PE) film with pendant long-chainpolystyrene (PS) grafts (molecular weight on the order of 10⁶, (seeBerg, et al., J. Am. Chem. Soc., 1989, 111, 8024 and InternationalPatent Application WO 90/02749),). The loading capacity of the film isas high as that of a beaded matrix with the additional flexibility toaccommodate multiple syntheses simultaneously. The PEPS film may befashioned in the form of discrete, labeled sheets, each serving as anindividual compartment. During all the identical steps of the syntheticcycles, the sheets are kept together in a single reaction vessel topermit concurrent preparation of a multitude of peptides at a rate closeto that of a single peptide by conventional methods. Also, experimentswith other geometries of the PEPS polymer such as, for example,non-woven felt, knitted net, sticks or microwell plates have notindicated any limitations of the synthetic efficacy.

Further support media amenable to the present invention include withoutlimitation particles based upon copolymers of dimethylacrylamidecross-linked with N,N′-bisacryloylethylenediamine, including a knownamount ofN-tertbutoxycarbonyl-beta-alanyl-N′-acryloylhexamethylenediamine.Several spacer molecules are typically added via the beta alanyl group,followed thereafter by the amino acid residue subunits. Also, the betaalanyl-containing monomer can be replaced with an acryloyl safcosinemonomer during polymerization to form resin beads. The polymerization isfollowed by reaction of the beads with ethylenediamine to form resinparticles that contain primary amines as the covalently linkedfunctionality. The polyacrylamide-based supports are relatively morehydrophilic than are the polystyrene-based supports and are usually usedwith polar aprotic solvents including dimethylformamide,dimethylacetamide, N-methylpyrrolidone and the like (see Atherton, etal., J. Am. Chem. Soc., 1975, 97, 6584, Bioorg. Chem. 1979, 8, 351, andJ. C. S. Perkin 1538 (1981)).

Other support media amenable to the present invention include withoutlimitation a composite of a resin and another material that is alsosubstantially inert to the organic synthesis reaction conditionsemployed. One exemplary composite (see Scott, et al., J. Chrom. Sci.,1971, 9, 577) utilizes glass particles coated with a hydrophobic,cross-linked styrene polymer containing reactive chloromethyl groups,and is supplied by Northgate Laboratories, Inc., of Hamden, Conn., USA.Another exemplary composite contains a core of fluorinated ethylenepolymer onto which has been grafted polystyrene (see Kent andMerrifield, Israel J. Chem. 1978, 17, 243 and van Rietschoten inPeptides 1974, Y. Wolman, Ed., Wiley and Sons, New York, 1975, pp.113-116). Contiguous solid supports other than PEPS, such as cottonsheets (Lebl and Eichler, Peptide Res. 1989, 2, 232) andhydroxypropylacrylate-coated polypropylene membranes (Daniels, et al.,Tetrahedron Lett. 1989, 4345). Acrylic acid-grafted polyethylene-rodsand 96-microtiter wells to immobilize the growing peptide chains and toperform the compartmentalized synthesis. (Geysen, et al., Proc. Natl.Acad. Sci. USA, 1984, 81, 3998). A “tea bag” containingtraditionally-used polymer beads. (Houghten, Proc. Natl. Acad. Sci. USA,1985, 82, 5131). Simultaneous use of two different supports withdifferent densities (Tregear, Chemistry and Biology of Peptides, J.Meienhofer, ed., Ann Arbor Sci. Publ., Ann Arbor, 1972 pp. 175-178).Combining of reaction vessels via a manifold (Gorman, Anal. Biochem.,1984, 136, 397). Multicolumn solid-phase synthesis (e.g., Krchnak, etal., Int. J. Peptide Protein Res., 1989, 33, 209), and Holm and Meldal,in “Proceedings of the 20th European Peptide Symposium”, G. Jung and E.Bayer, eds., Walter de Gruyter & Co., Berlin, 1989 pp. 208-210).Cellulose paper (Eichler, et al., Collect. Czech. Chem. Commun., 1989,54, 1746). Support mediated synthesis of peptides have also beenreported (see, Synthetic Peptides: A User's Guide, Gregory A. Grant, Ed.Oxford University Press 1992; U.S. Pat. Nos. 4,415,732; 4,458,066;4,500,707; 4,668,777; 4,973,679; 5,132,418; 4,725,677 and Re-34,069.)

Equipment for Synthesis

Commercially available equipment routinely used for the support mediabased synthesis of oligomeric compounds and related compounds is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. Suitable solid phasetechniques, including automated synthesis techniques, are described inF. Eckstein (ed.), Oligonucleotides and Analogues, a Practical Approach,Oxford University Press, New York (1991).

Phosphorus Protecting Groups

In general, the phosphorus protecting group (pg) is an alkyl group or aβ-eliminable group having the formula —CH₂CH₂-G_(w), wherein G_(w) is anelectron-withdrawing group. Suitable examples of pg that are amenable touse in connection with the present invention include those set forth inthe Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777;4,973,679; and 5,132,418; and Koster U.S. Pat. No. 4,725,677 and Re.34,069. In general the alkyl or cyanoethyl withdrawing groups arepreferred, as commercially available phosphoramidites generallyincorporate either the methyl or cyanoethyl phosphorus protecting group.

The method for removal of phosphorus protecting groups (pg's) dependsupon the specific pg to be removed. The β-eliminable groups, such asthose disclosed in the Köster et al. patents, are generally removed in aweak base solution, whereby an acidic β-hydrogen is extracted and the—CH₂CH₂-G_(w) group is eliminated by rearrangement to form thecorresponding acrylo-compound CH₂═CH-G_(w). In contrast, an alkyl groupis generally removed by nucleophilic attack on the α-carbon of the alkylgroup. Such pg's are described in the Caruthers et al. patents, as citedherein.

Oxidation (Including Sulfurization)

The person skilled in the art will recognize that oxidation of P(III) toP(V) can be carried out by a variety of reagents. Furthermore, theperson skilled in the art will recognize that the P(V) species can existas phosphate triesters, phosphorothioate diesters, or phosphorodithioatediesters. Each type of P(V) linkage has uses and advantages, asdescribed herein. Thus, the term “oxidizing agent” should be understoodbroadly as being any reagent capable of transforming a P(III) species(e.g. a phosphite) into a P(V) species. Thus the term “oxidizing agent”includes “sulfurizing agent,” and oxidation will be understood toembrace both introduction of oxygen and introduction of sulfur, orsulfurization. Where it is important to indicate that an oxidizing agentintroduces an oxygen into a P(III) species to make a P(V) species, theoxidizing agent will be referred to herein is “an oxygen-introducingoxidizing reagent.”

Oxidizing reagents for making phosphate diester linkages (i.e.oxygen-introducing oxidizing reagents) under the phosphoramiditeprotocol have been described by e.g. Caruthers et al. and Köster et al.,as cited herein. Examples of sulfurization reagents which have been usedto synthesize oligonucleotides containing phosphorothioate bonds includeelemental sulfur, dibenzoyltetrasulfide, 3-H-1,2-benzidithiol-3-one1,1-dioxide (also known as Beaucage reagent), tetraethylthiuramdisulfide (TETD), and bis O,O-diisopropoxy phosphinothioyl) disulfide(known as Stec reagent). Oxidizing reagents for making phosphorothioatediester linkages include phenyl acetyl disulfide (PADS), as described byCole et al. in U.S. Pat. No. 6,242,591. In some embodiments of theinvention, the phosphorothioate diester and phosphate diester linkagesmay alternate between sugar subunits. In other embodiments of thepresent invention, phosphorothioate linkages alone may be employed.

Various solvents may be used in the oxidation reaction. Suitablesolvents are identified in the Caruthers et al. and Koster et al.patents, cited herein. The Cole et al. patent describes acetonitrile asa solvent for phenyl acetyl disulfide. Other suitable solvents includetoluene, xanthenes, dichloromethane, etc.

Cleavage and Workup

Reagents for cleaving an oligonucleotide from a support are set forth,for example, in the Caruthers et al. and Koster et al. patents, as citedherein.

The oligonucleotide may be worked up by standard procedures known in theart, for example by size exclusion chromatography, high performanceliquid chromatography (e.g. reverse-phase HPLC), differentialprecipitation, etc. In some embodiments according to the presentinvention, the oligonucleotide is cleaved from a solid support while the5′-OH protecting group is still on the ultimate nucleoside. Thisso-called DMT-on (or trityl-on) oligonucleotide is then subjected tochromatography, after which the DMT group is removed by treatment in anorganic acid, after which the oligonucleotide is de-salted and furtherpurified to form a final product.

5′-Deprotection

In general, the 5′-hydroxylprotecting groups may be any groups that areselectively removed under suitable conditions. In particular, the4,4′-dimethoxytriphenylmethyl (DMT) group is a favored group forprotecting at the 5′-position, because it is readily cleaved underacidic conditions (e.g. in the presence of dichloroacetic acid (DCA),trichloroacetic acid (TCA), or acetic acid. Removal of DMT from thesupport-bound oligonucleotide is generally performed with DCA. Inembodiments of the present invention, at least one of the 5′-protectinggroups is DMT and the reagent for removing the 5′-protecting group fromthat nucleotide is DCA. In some embodiments of the invention, aplurality of the 5′-protecting groups is DMT, and the reagents forremoving those protecting groups are all DCA, optionally in a suitablesolvent, such as acetonitrile or toluene. In still other embodiments ofthe invention, each of the 5′-protecting group is DMT, and all but thefinal DMT group is removed on the support using DCA in a suitablesolvent as deprotecting reagent, the final DMT group being removed afterthe oligonucleotide has been cleaved from the support, as describedabove. In some other embodiments of the invention, each 5′-protectinggroup is DMT and each 5′-protecting group is removed using DCA while theoligonucleotide is on the solid support.

In some embodiments of the present invention, the last 5′-protectinggroup may be other than DMT, e.g. pixyl, and the final 5′-protectinggroup may be removed using an acid other than DCA.

Removal of 5-protection after cleavage of the oligonucleotide from thesupport is generally performed with acetic acid, however a weaker acidmay be used in the case of more labile protecting groups than DMT.

Oligomer Design Considerations

In naturally occurring oligonucleotides, the sugar ring is β-D-ribosyl(RNA) or β-D-2′-deoxyribosyl (DNA). The hybridization behavior of DNAwith RNA differs from the hybridization of RNA to RNA. This differencegives rise to different in vitro and in vivo effects. For example,DNA-RNA hybrids effectively bind to RNAse H, which results in scissionof RNA. In contrast, RNA-RNA hybrids may be unwound by helicase, wherebythe antisense strand is permitted to form a hybrid with mRNA. Theexogenous RNA-mRNA hybrid interacts with one or more members of the RISCcomplex, which effects mRNA scission.

Synthetic sugars and sugar analogs are designed to adopt certain spatialconformations that resemble DNA, RNA or some structure intermediatebetween these conformations. Again, the sugar or sugar analog functionsas a sort of platform to hold the base in the correct orientation tointeract with bases on the opposite strand. The sugar or sugar analog(collectively skeletal members) also provides binding sites for thelinking groups, which join the monomeric units together to form theoligomer. The conformation of the sugar or sugar analog greatlyinfluences the spatial orientations of the bases and linking groups, andalso greatly influences the shape of the antisense-sense hybrid insolution. This conformational influence can have an important impact onthe efficacy of the antisense compound in modulation of gene expression.

In the broadest sense, the term “oligonucleotide” refers to an oligomerhaving a plurality of skeletal members, e.g. sugar units (ribosyl,deoxyribosyl, arabinosyl, modified sugar unit, etc.) linked by phosphatediester linkers (i.e. phosphoryl or thiophosphoryl diester), and havingbases for establishing binding to complementary oligomer strands. Insome embodiments of the invention, an oligonucleotide may contain bothphosphoryl diester and phosphorothioate linkers. In other embodiments,the linkers are all phosphorothioate linkers. While phosphoryl linkersare the naturally occurring type of linkers in oligonucleotides,thiophosphate linkers are known to confer nuclease stability tooligonucleotides cells. Hence, it is often preferred to prepareoligonucleotides with at least a portion of the phosphate diestermoieties replaced by phosphorothioate diester moieties.

As described herein, oligonucleotides can be prepared as chimeras withother oligomeric moieties. In the context of this invention, the term“oligomeric compound” refers to a polymeric structure capable ofhybridizing a region of a nucleic acid molecule, and an “oligomericmoiety” a portion of such an oligomeric compounds. Oligomeric compoundsinclude oligonucleotides, oligonucleosides, oligonucleotide analogs,modified oligonucleotides and oligonucleotide mimetics. Oligomericcompounds can be linear or circular, and may include branching. They canbe single stranded or double stranded, and when double stranded, mayinclude overhangs. In general an oligomeric compound comprises abackbone of linked monomeric subunits where each linked monomericsubunit is directly or indirectly attached to a heterocyclic basemoiety. The linkages joining the monomeric subunits, the monomericsubunits and the heterocyclic base moieties can be variable in structuregiving rise to a plurality of motifs for the resulting oligomericcompounds including hemimers, gapmers and chimeras. As is known in theart, a nucleoside is a base-sugar combination. The base portion of thenucleoside is normally a heterocyclic base moiety. The two most commonclasses of such heterocyclic bases are purines and pyrimidines. In thecontext of this invention, the term “oligonucleoside” refers tonucleosides that are joined by internucleoside linkages that do not havephosphorus atoms. Internucleoside linkages of this type include shortchain alkyl, cycloalkyl, mixed heteroatom alkyl, mixed heteroatomcycloalkyl, one or more short chain heteroatomic and one or more shortchain heterocyclic. These internucleoside linkages include but are notlimited to siloxane, sulfide, sulfoxide, sulfone, acetyl, formacetyl,thioformacetyl, methylene formacetyl, thioformacetyl, alkeneyl,sulfamate; methyleneimino, methylenehydrazino, sulfonate, sulfonamide,amide and others having mixed N, O, S and CH₂ component parts.

Uses for Oligomers

Oligomers, and especially oligonucleotides and chimeras according to thepresent invention, have been used in a variety of applications,including in assays, sequence arrays, primers and probes for nucleicacid amplification (e.g. PCR), as antisense molecules for gene targetvalidation and therapeutic applications, etc. The person skilled in theart will recognize understand that the methods according to the presentinvention may be adapted to prepare oligomers for such applications.Accordingly, only select uses of oligomers according to the presentinvention will be described herein.

Antisense

Exemplary preferred antisense compounds include DNA or RNA sequencesthat comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred antisense compounds(the remaining nucleobases being a consecutive stretch of the same DNAor RNA beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid aid continuing until the DNA or RNA contains about 8 toabout 80 nucleobases). Similarly preferred antisense compounds arerepresented by DNA or RNA sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same DNA or RNA beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the DNA or RNA contains about 8 to about 80 nucleobases). Onehaving skill in the art, once armed with the empirically-derivedpreferred antisense compounds illustrated herein will be able, withoutundue experimentation, to identify further preferred antisensecompounds.

Antisense and other compounds of the invention, which hybridize to thetarget and inhibit expression of the target, are identified throughexperimentation, and representative sequences of these compounds areherein identified as preferred embodiments of the invention. Whilespecific sequences of the antisense compounds are set forth herein, oneof skill in the art will recognize that these serve to illustrate anddescribe particular embodiments within the scope of the presentinvention. Additional preferred antisense compounds may be identified byone having ordinary skill.

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Research Reagents, Diagnostics, Kits

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

For use in kits and diagnostics, the antisense compounds of the presentinvention, either alone or in combination with other antisense compoundsor therapeutics, can be used as tools in differential and/orcombinatorial analyses to elucidate expression patterns of a portion orthe entire complement of genes expressed within cells and tissues.

Expression patterns within cells or tissues treated with one or moreantisense compounds are compared to control cells or tissues not treatedwith antisense compounds and the patterns produced are analyzed fordifferential levels of gene expression as they pertain, for example, todisease association, signaling pathway, cellular localization,expression level, size, structure or function of the genes examined.These analyses can be performed on stimulated or unstimulated cells andin the presence or absence of other compounds which affect expressionpatterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression)(Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (reviewed in To, Comb. Chem. High Throughput Screen, 2000, 3,235-41).

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat oligonucleotides can be useful therapeutic modalities that can beconfigured to be useful in treatment regimes for treatment of cells,tissues and animals, especially humans.

It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding a particular protein. The targeting process alsoincludes determination of a site or sites within this gene for theantisense interaction to occur such that the desired effect, e.g.,detection or modulation of expression of the protein, will result.Within the context of the present invention, a preferred intragenic siteis the region encompassing the translation initiation or terminationcodon of the open reading frame (ORF) of the gene. Since, as is known inthe art, the translation initiation codon is typically 5′-AUG (intranscribed mRNA molecules; 5′-ATG in the corresponding DNA molecule),the translation initiation codon is also referred to as the “AUG codon,”the “start codon” or the “AUG start codon”. A minority of genes have atranslation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function invivo. Thus, the terms “translation initiation codon” and “start codon”can encompass many codon sequences, even though the initiator amino acidin each instance is typically methionine (in eukaryotes) orformylmethionine (in prokaryotes). It is also known in the art thateukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areusedin vivo to initiate translation of an mRNA molecule transcribed froma gene encoding a particular protein, regardless of the sequence(s) ofsuch codons.

It is also known in the art that a translation termination codon (or“stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA,5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAGand 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Other target regions include the 5′ untranslatedregion (5′UTR), known in the art to refer to the portion of an mRNA inthe 5′ direction from the translation initiation codon, and thusincluding nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. mRNA splice sites, i.e., intron-exonjunctions, may also be preferred target regions, and are particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular mRNA splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred targets. mRNA transcripts produced viathe process of splicing of two (or more) mRNAs from different genesources are known as “fusion transcripts”. It has also been found thatintrons can be effective, and therefore preferred, target regions forantisense compounds targeted, for example, to DNA or pre-mRNA.

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic andextronic regions.

Upon excision of one or more exon or intron regions or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through theuse of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites.

Once one or more target sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired effect.

In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable.

An antisense compound is specifically hybridizable when binding of thecompound to the target DNA or RNA molecule interferes with the normalfunction of the target DNA or RNA to cause a loss of activity, and thereis a sufficient degree of complementarity to avoid nonspecific bindingof the antisense compound to non-target sequences under conditions inwhich specific binding is desired, i.e., under physiological conditionsin the case of in vivo assays or therapeutic treatment, and in the caseof in vitro assays, under conditions in which the assays are performed.It is preferred that the antisense compounds of the present inventioncomprise at least 80% sequence complementarity with the target nucleicacid, more that they comprise 90% sequent complementarity and even morecomprise 95% sequence complementarity with the target nucleic acidsequence to which they are targeted. Percent complementarity of anantisense compound with a target nucleic acid can be determinedroutinely using basic local alignment search tools (BLAST programs)(Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden,Genome Res., 1997, 7, 649-656).

Antisense and other compounds of the invention, which hybridize to thetarget and inhibit expression of the target, are identified throughexperimentation, and representative sequences of these compounds arehereinbelow identified as preferred embodiments of the invention. Thesites to which these preferred antisense compounds are specificallyhybridizable are hereinbelow referred to as “preferred target regions”and are therefore preferred sites for targeting. As used herein the term“preferred target region” is defined as at least an 8-nucleobase portionof a target region to which an active antisense compound is targeted.While not wishing to be bound by theory, it is presently believed thatthese target regions represent regions of the target nucleic acid whichare accessible for hybridization.

While the specific sequences of particular preferred target regions areset forth below, one of skill in the art will recognize that these serveto illustrate and describe particular embodiments within the scope ofthe present invention. Additional preferred target regions may beidentified by one having ordinary skill.

Target regions 8-80 nucleobases in length comprising a stretch of atleast eight (8) consecutive nucleobases selected from within theillustrative preferred target regions are considered to be suitablepreferred target regions as well.

Exemplary good preferred target regions include DNA or RNA sequencesthat comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred target regions (theremaining nucleobases being a consecutive stretch of the same DNA or RNAbeginning immediately upstream of the 5′-terminus of the target regionand continuing until the DNA or RNA contains about 8 to about 80nucleobases). Similarly good preferred target regions are represented byDNA or RNA sequences that comprise at least the 8 consecutivenucleobases from the 3′-terminus of one of the illustrative preferredtarget regions (the remaining nucleobases being a consecutive stretch ofthe same DNA or RNA beginning immediately downstream of the 3′-terminusof the target region and continuing until the DNA or RNA contains about8 to about 80 nucleobases). One having skill in the art, once armed withthe empirically-derived preferred target regions illustrated herein willbe able, without undue experimentation, to identify further preferredtarget regions. In addition, one having ordinary skill in the art willalso be able to identify additional compounds, including oligonucleotideprobes and primers, that specifically hybridize to these preferredtarget regions using techniques available to the ordinary practitionerin the art.

The ability of oligonucleotides to bind to their complementary targetstrands is compared by determining the melting temperature (T_(m)) ofthe hybridization complex of the oligonucleotide and its complementarystrand. The melting temperature (T_(m)), a characteristic physicalproperty of double helices, denotes the temperature (in degreescentigrade) at which 50% helical (hybridized) versus coil (unhybridized)forms are present. T_(m) is measured by using the UV spectrum todetermine the formation and breakdown (melting) of the hybridizationcomplex. Base stacking, which occurs during hybridization, isaccompanied by a reduction in UV absorption (hypochromicity).Consequently, a reduction in UV absorption indicates a higher T_(m). Thehigher the T_(m), the greater the strength of the bonds between thestrands. The structure-stability relationships of a large number ofnucleic acid modifications have been reviewed (Freier and Altmann, Nucl.Acids Research, 1997, 25, 4429-443).

The person having skill in the art will recognize that furtherembodiments are possible within the general scope of the foregoingdescription and the attached drawings and claims, and it would be withinthe skill of such skilled person to practice the invention as generallydescribed herein.

All references cited herein, including all patents, patent documents,applications, published application, and non-patent references, areexpressly incorporated herein by reference.

1. A process comprising contacting an oligonucleotide withdichloroacetic acid in the substantial absence of chloral impurity. 2.The process of claim 1, wherein said oligonucleotide bears a blockinggroup.
 3. The process of claim 2, wherein said contacting is performedfor a time and under conditions effective to remove said blocking group.4. The process of claim 2, wherein said blocking group is a tritylgroup.
 5. The process of claim 1, wherein said dichloroacetic acid ispresent in the form of a dichloroacetic acid-containing solution.
 6. Theprocess of claim 5, wherein said solution comprises less than about 0.03weight percent chloral hydrate.
 7. An oligonucleotide prepared by theprocess of claim
 2. 8. The oligonucleotide of claim 7, wherein theoligonucleotide adducts of chloral are excluded to the extent that theirconcentration is below the limit of detection for oligonucleotideadducts of chloral.
 9. A process, comprising: subjecting dichloroaceticacid comprising an initial concentration of chloral impurity todistillation; and collecting at least one fraction of dichloroaceticacid, said fraction having a lower concentration of chloral impurityrelative to said initial concentration.
 10. The process of claim 9,wherein the distillation is vacuum distillation.
 11. The process ofclaim 9, wherein said lower concentration of chloral impurity is belowabout 0.03 weight percent.
 12. The process of claim 11, wherein thelower concentration of chloral impurity is below the limit of detectionfor chloral impurity.
 13. The process of claim 9, further comprisingcontacting a blocked hydroxyl group of an oligonucleotide with said atleast one fraction of dichloroacetic acid fraction under conditionssuitable to remove said blocked hydroxyl group.
 14. The process of claim13, wherein the lower concentration of chloral impurity is below about0.03 weight percent.
 15. The process of claim 14, wherein the lowerconcentration of chloral impurity is below the limit of detection forchloral impurity.
 16. The process of claim 13, wherein said blockedhydroxyl group comprises a 4,4′-dimethoxytriphenylmethyl blocking group.17. The process of claim 13, wherein said blocked hydroxyl groupcomprises a blocked 5′-hydroxyl group.
 18. The process of claim 13,wherein said distillation comprises vacuum distillation.
 19. The processof claim 13, whereby an oligonucleotide substantially free of chloraladducts is obtained.
 20. The process of claim 9, wherein said chloralimpurity comprises chloral hydrate.